Production of fertile XY animals from XY ES cells

ABSTRACT

Methods and compositions are described for making phenotypically female fertile animals from XY donor cells and suitable host embryos. Culture media and methods are provided for maintaining XY donor cells in culture that after introduction into a host embryo and gestation in a suitable host will result in fertile XY female animals. Methods and compositions are described for making fertile female animals in an F0 generation from a donor XY cell and a host embryo, as are methods for making F1 progeny that are homozygous for a modification from a heterozygous F0 fertile male and a heterozygous F0 fertile female sibling.

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 61/353,896, filed on Jun. 11, 2010.

FIELD

The invention relates to the manufacture of fertile female animalsderived from XY embryonic stem (“ES”) cells and having a XY karyotype.In a particular embodiment, methods and compositions for making fertileXY female mice from XY donor murine ES cells are described. In vitrofertilization methods for favoring the formation of phenotypic femalesare also described.

BACKGROUND

Nearly all commonly employed ES cell lines for making geneticallymodified mice are genotypic male (XY) ES cell lines. As a result, in theF0 generation, all XY animals are male. Most genetic modifications arecarried out by targeting the XY ES cells to create a modification of oneof two existing alleles, i.e., the donor mouse ES cell is heterozygousfor the genetic modification. However, it is often desirable to obtain amouse that is homozygous for the genetic modification. Becauseessentially no fully ES cell-derived female mice are born in the F0generation that comprise the modification, the F0 male is typically bredto a female (e.g., a matched inbred female) to generate a litter inwhich at least one female (an F1 female) might be heterozygous for thegenetic modification. The heterozygous F1 female is then intercrossedwith an F1 heterozygous male, to obtain a homozygous progeny. Suchbreeding requirements represent costly and time-consuming steps. It isdesirable to generate a breeding pair in an F0 generation, or at leastto generate an F0 female that is largely or fully derived from the donor(XY male) ES cell.

There is a need in the art for methods and compositions for making afertile female animal in the F0 generation from a donor male (XY) EScell and a host embryo.

SUMMARY

In one aspect, a method for making a fertile female nonhuman animal froman XY donor cell is provided, comprising: (a) introducing a nonhuman XYdonor cell into a nonhuman host embryo to form a chimeric embryo; and,(b) gestating the chimeric embryo to form a nonhuman female animal,wherein the nonhuman female animal is XY and upon attaining sexualmaturity is fertile.

In one embodiment, the nonhuman animal is a mouse.

In one embodiment, the nonhuman XY female animal is formed in the F0generation. In one embodiment, the nonhuman female XY animal in the F0generation is a mouse and has a coat color 100% derived from the donorcell. In one embodiment, the nonhuman female XY animal formed in the F0generation is at least 90%, 92%, 94%, 96%, 98%, or 99.8% derived fromthe XY donor cell. In one embodiment, the nonhuman female XY animal inthe F0 generation is about 100% derived from the donor cell. In oneembodiment, the contribution of a host embryo cell to the nonhumanfemale XY animal in the F0 generation is determined by a quantitativeassay that is capable of detecting 1 cell in 2,000 (0.05%), and notissue of the female XY animal is positive for host embryo cellcontribution.

In one embodiment, the donor cell comprises a genetic modification. Inone embodiment, the genetic modification comprises a deletion in wholeor in part of an endogenous nucleic acid sequence; a substitution of oneor more nucleic acids; a replacement of an endogenous nucleic acidsequence, e.g. a gene, in whole or in part with a heterologous nucleicacid sequence; a knockout; and/or, a knock-in.

In one embodiment, the method further comprises a step of breeding an F0generation XY male heterozygous for the genetic modification with a F0generation XY female heterozygous for the genetic modification (e.g., asibling), and obtaining from said breeding an F1 generation animalhomozygous for the genetic modification.

In one embodiment, the XY donor cell before introduction into the hostembryo is maintained in a medium comprising base medium and supplements,wherein the base medium exhibits a characteristic selected from thegroup consisting of: (a) an osmolality of about 250-310 mOsm/kg; (b) aconductivity of about 11-13 mS/cm; (c) an alkaline metal and halide saltin a concentration of about 60-105 mM; (d) a carbonic acid saltconcentration of about 20-30 mM; (e) a total alkaline metal halide saltand carbonic acid salt concentration of no more than about 85-130 mM;and (f) a combination thereof.

In one embodiment, the supplements comprise components for maintainingES cells in culture. In one embodiment, the supplements comprise one ormore of fetal bovine serum (FBS), glutamine, antibiotic(s), pyruvate,nonessential amino acids, 2-mercaptoethanol, and LIF.

In one embodiment, the base medium is a low-salt DMEM. In a specificembodiment, the low-salt DMEM has an NaCl concentration of 85-130 mM. Inone embodiment, the base medium is a low osmolality DMEM. In a specificembodiment, the low osmolality DMEM has an osmolality of 250-310mOsm/kg. In one embodiment, the base medium is a low conductivity DMEM.In a specific embodiment, the low conductivity DMEM has a conductivityof 11-13 mS/cm.

In one embodiment, the donor cell is maintained in the recited basemedium plus supplements before introduction into the host embryo forabout 1, 2, 3, 4, 5, 6 days, 1 week, 8, 9, 110, 11, or 12 days, 2 weeks,3 weeks, or 4 weeks or more. In a specific embodiment, the donor cell ismaintained in the base medium plus supplements for at least a weekbefore introduction into the host embryo. In a specific embodiment, thedonor cell is maintained in the base medium plus supplements for 2-4weeks before introduction into the host embryo.

In one embodiment, the host embryo is a 2-cell stage, 4-cell stage,8-cell stage, 16-cell stage, 32-cell stage, or 64-cell stage embryo. Inanother embodiment, the host embryo is a blastocyst. In one embodiment,the embryo is in a stage selected from a pre-morula stage, a morulastage, an uncompacted morula stage, and a compacted morula stage. In oneembodiment, the embryo stage is selected from a Theiler Stage 1 (TS1), aTS2, a TS3, a TS4, a TS5, and a TS6, with reference to the Theilerstages described in Theiler (1989) “The House Mouse: Atlas of MouseDevelopment,” Springer-Verlag, N.Y. In a specific embodiment, theTheiler Stage is selected from TS1, TS2, TS3, and a TS4. In oneembodiment, the embryo comprises a zona pellucida, and the donor cell isan ES cell that is introduced into the embryo through a hole in the zonapellucida.

In one embodiment, the embryo comprises a pre-blastocyst embryo. In oneembodiment, the embryo is a morula-stage embryo. In a specificembodiment, the morula-stage embryo is aggregated. In one embodiment,the embryo is a zona-less embryo.

In one embodiment, the XY donor cell is selected from an ES cell, aninduced pluripotent stem (iPS) cell, a pluripotent cell, and atotipotent cell. In a specific embodiment, the XY donor cell is a mouseES cell and the host embryo is a mouse embryo.

In one embodiment, the XY donor cell is an ES cell from an inbred mousestrain. In one embodiment, the XY donor cell is an ES cell from a hybridor outbred mouse strain.

In one embodiment, the host embryo is a mouse host embryo. In oneembodiment, the mouse host embryo is from an inbred strain, in anotherembodiment from a hybrid or an outbred strain. In one embodiment, thedonor cell is a mouse donor cell. In one embodiment, the host embryo andthe donor cell are both mouse, and each is independently selected from amouse that is a 129 strain, a C57BL/6 strain, a mix of 129 and C57BL/6,a BALB/c strain, or a Swiss Webster strain. In a specific embodiment,the mouse is 50% 129 and 50% C57BL/6. In one embodiment, the mouse is a129 strain selected from the group consisting of a strain that is 129P1,129P2, 129P3, 129X1, 129S1 (e.g., 12951/SV, 12951/SvIm), 129S2, 129S4,129S5, 12959/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2(see, e.g., Festing et al. (1999) Revised nomenclature for strain 129mice, Mammalian Genome 10:836). In one embodiment the mouse is a C57BLstrain, in a specific embodiment selected from C57BL/A, C57BL/An,C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ,C57BL/10, C57BL/10ScSn, C57BL/10Cr, C57BL/Ola. In a specific embodiment,the mouse is a mix of an aforementioned 129 strain and an aforementionedC57BL/6 strain. In another specific embodiment, the mouse is a mix ofaforementioned 129 strains, or a mix of aforementioned BL/6 strains. Ina specific embodiment, the 129 strain of the mix is a 129S6(129/SvEvTac) strain.

In one embodiment, the XY female mouse produces 1, 2, 3, 4, 5, 6, 7, 8,or 9 litters of live mice during its lifetime. In one embodiment, the XYfemale mouse produces at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pups perlitter. In one embodiment, the XY female mouse produces about 4-6 pupsper litter. In one embodiment, the XY female mouse produces 2-6 litters,wherein each litter has at least 2, 3, 4, 5, or 6 pups. In oneembodiment, about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of thepups are XY female pups. In a specific embodiment, about 15%-25% are XYfemale pups.

In one aspect, a method for making a mouse that is homozygous for agenetic modification is provided, employing an XY ES cell that isheterozygous for the genetic modification. In one embodiment, the methodcomprises genetically modifying an XY donor ES cell to form aheterozygous XY donor ES cell, maintaining the heterozygous XY donor EScell in a low salt and/or low osmolality or low conductivity medium,introducing the heterozygous XY donor ES cell into a pre-morula hostembryo, gestating the host embryo, after gestation obtaining a fertileF0 generation female XY mouse that comprises the heterozygousmodification and is at least in part derived from the donor ES cell, andafter gestation obtaining a fertile F0 generation male XY mouse thatcomprises the heterozygous modification and that is at least in partderived from the donor ES cell, and breeding the F0 male and the F0female to obtain an F1 progeny that comprises a homozygous modification.

In one embodiment, the F0 generation female XY mouse and/or the F0generation male XY mouse is at least 20% or more derived from the donorES cell. In one embodiment, the F0 female XY mouse is at least 30%, 40%,50%, 60%, 70%, or 80% derived from the donor ES cell.

In one embodiment, the F0 generation female XY mouse and/or the male XYmouse is at least 90% derived from the donor ES cell. In one embodiment,the F0 generation female XY mouse is at least 92%, 94%, 96%, 98%, 99%,or 99.8% derived from the donor ES cell. In one embodiment, the F0female XY mouse and/or the F0 male XY mouse has a coat color that is100% derived from the ES cell.

In one embodiment, the F0 generation mouse comprises an XY oocyte.

In one embodiment, the F1 generation progeny mouse comprises a genomecompletely derived from the donor ES cell.

In one embodiment, the frequency of crosses of F0 generation male and F0generation female mice that give rise to fully ES cell-derived mice is100%.

In one aspect, a method for generating a mouse pup litter is provided,comprising introducing XY donor ES cells prepared according to theinvention into host mouse embryos, gestating the embryos in a suitablemouse, and obtaining a litter of mouse pups that comprises at least oneXY female mouse pup that upon reaching sexual maturity is a fertile XYfemale mouse.

In one embodiment, the percentage of XY female mouse pups born that uponreaching sexual maturity are fertile is about 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, or 50%. In a specific embodiment, the percentage is about15-25%.

In one aspect, a method for maintaining an XY ES cell in culture isprovided, wherein the XY ES cell is maintained under conditions thatpromote or favor development of a female XY mouse following introductionof the XY ES cell into a host embryo and following gestation in asuitable female mouse. The method comprises maintaining the male ES cellin a suitable culture medium that comprises a base medium andsupplements, wherein the base medium exhibits an osmolality of about240-320 mOsm/kg, a conductivity of about 10-14 mS/cm, an alkaline metalhalide salt concentration of about 50-105 mM, a salt of carbonic acidconcentration of 10-40 mM, and/or a combined alkaline metal salt andcarbonic acid salt concentration of about 80-140 mM. In one embodiment,the XY ES cell is maintained in the medium (with supplements formaintaining ES cells) for a period of 1, 2, 3, 4, 5, or 6 days, or 1week, 8, 9, 110, 11, or 12 days, 2 weeks, 3 weeks, or 4 weeks prior tointroduction into a host embryo. In a specific embodiment, the ES cellis maintained in the medium (low-salt base medium with supplements formaintaining ES cells) for about 2-4 weeks prior to introduction into thehost embryo.

In one embodiment, the base medium exhibits an osmolality of no morethan about 320, 310, 300, 290, 280, 270, 260, 250, or 240 mOsm/kg. Inone embodiment, the base medium exhibits an osmolality of no more thanabout 240-320, 250-310, or 260-300 mOsm/kg. In a specific embodiment,the base medium exhibits an osmolality of about 270 mOsm/kg.

In one embodiment, the base medium exhibits a conductivity of no morethan about 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, or 14.0mS/cm. In one embodiment, the base medium exhibits a conductivity of nomore than about 10-14 mS/cm or 11-13 mS/cm. In a specific embodiment,the base medium exhibits a conductivity of about 12-13 mS/cm.

In a specific embodiment, the base medium exhibits a conductivity ofabout 12-13 mS/cm and an osmolality of about 260-300 mOsm/kg. In afurther specific embodiment, the base medium comprises sodium chlorideat a concentration of about 90 mM NaCl. In a further specificembodiment, the concentration of sodium chloride is about 70-95 mM. In afurther specific embodiment, the base medium comprises sodiumbicarbonate at a concentration of less than about 35 mM. In a furtherspecific embodiment, the concentration of sodium bicarbonate is about20-30 mM.

In one embodiment, the base medium exhibits a concentration of a salt ofan alkaline metal and a halide of no more than about 100 mM. In oneembodiment, the salt of the alkaline metal and the halide is NaCl. Inone embodiment, the concentration of the salt of the alkaline metal andhalide is no higher than 90, 80, 70, 60, or 50 mM. In one embodiment,the concentration in the base medium of the salt of the alkaline metaland halide is about 60-105, 70-95, or 80-90 mM. In a specificembodiment, the concentration is about 85 mM.

In one embodiment, the base medium exhibits a concentration of a salt ofcarbonic acid. In one embodiment, the salt of carbonic acid is a sodiumsalt. In one embodiment, the sodium salt is sodium bicarbonate. In oneembodiment, the concentration of carbonic acid salt in the base mediumis no higher than 40, 35, 30, 25, or 20 mM. In one embodiment theconcentration of carbonic acid salt in the base medium is about 10-40,in another embodiment about 20-30 mM. In a specific embodiment, theconcentration is about 25 or 26 mM.

In one embodiment, the sum of the concentration of the salt of thealkaline metal and halide and the salt of carbonic acid in the basemedium is no more than 140, 130, 120, 110, 100, 90, or 80 mM. In oneembodiment, the sum of the concentration of the salt of the alkalinemetal and halide and the salt of carbonic acid in the base medium isabout 80-140, 85-130, 90-120, 95-120, or 100-120 mM. In a specificembodiment, the sum of the concentration of the salt of the alkalinemetal and halide and the salt of carbonic acid in the base medium isabout 115 mM.

In one embodiment, the molar ratio of the salt of the alkaline metal andhalide and the salt of carbonic acid is higher than 2.5. In oneembodiment, the ratio is about 2.6-4.0, 2.8-3.8, 3-3.6, or 3.2-3.4. Inone embodiment, the ratio is 3.3-3.5. In a specific embodiment, theratio is 3.4.

In one embodiment, the base medium exhibits an osmolality of about250-310 mOsm/kg, and a concentration of a salt of an alkaline metal anda halide of about 60-105 mM. In a further embodiment, the base mediumhas a concentration of a salt of carbonic acid of about 20-30 mM. In afurther embodiment, the sum of the concentrations of the salt of analkaline metal and halide and the salt of carbonic acid is about 80-140mM. In a further embodiment, the conductivity of the base medium isabout 12-13 mS/cm.

In one aspect, a method for maintaining a donor XY ES cell in culture isprovided, under conditions as described herein, wherein followingintroduction of the donor XY ES cell into a host embryo to form achimeric embryo and gestation of the chimeric embryo in a suitableanimal, the chimeric embryo develops into a mouse pup that is at least90% XY and is a female which, upon attaining sexual maturity, isfertile.

In one embodiment, the mouse pup is at least 92%, 94%, 96%, 98%, or99.8% XY.

In one aspect, a method is provided for making a fertile XY femaleanimal, comprising maintaining an XY donor cell in a medium comprisinglow-salt base medium prior to introduction of the donor cell into a hostembryo, introducing the donor cell into the host embryo, gestating thehost embryo in a suitable animal to term, and following gestationobtaining an XY female animal therefrom, wherein upon reaching sexualmaturity the XY female animal is fertile.

In one embodiment, the XY donor cell is a mouse ES cell, and the hostembryo is an embryo from an XX female mouse.

In one embodiment, the culture in which the donor cell is maintainedcomprises a base medium as described herein, and one or more supplementssuitable for maintaining mouse ES cells in culture. In a specificembodiment, the one or more supplements suitable for maintaining a mouseES cell in culture are FBS (90 mL FBS/0.5 L base medium), glutamine (2.4mmoles/0.5 L base medium), sodium pyruvate (0.6 mmoles/0.5 L basemedium), nonessential amino acids (<0.1 mmol/0.5 L base medium),2-mercaptoethanol, LIF, and one or more antibiotics.

In one embodiment, the donor cell is maintained in a medium with alow-salt base medium for at least 1, 2, 3, 4, 5, or 6 days, or 1 week,8, 9, 110, 11, or 12 days, 2 weeks, 3 weeks, or 4 weeks prior tointroducing the donor cell into a host embryo. In a specific embodiment,the donor cell is maintained in a medium with a low-salt base medium atleast 2-4 weeks prior to introduction of the donor cell into the hostembryo.

In one embodiment, the donor cell is maintained (e.g., frozen) in amedium comprising low-salt base medium, and the donor cell is thawed inand maintained in the medium comprising low-salt base medium for atleast 1, 2, 3, or 4 or more days before introducing the donor cell intothe host embryo. In a specific embodiment, the donor cell is passaged atleast once in a medium comprising low-salt base medium, the cell isfrozen in the medium comprising low-salt base medium, and the cell isthawed in a medium comprising low-salt base medium and grown for 1, 2,3, 4, 5, or 6 days or more, or 1 week, 8, 9, 110, 11, or 12 days, 2weeks, 3 weeks, 4 weeks, or more prior to introduction into the hostembryo.

In one embodiment, the donor cell is maintained for a period of one,two, three, or four days prior to introduction into a host embryo. In onembodiment, the donor cell is maintained in the medium comprising therecited base medium for a period of 3 days.

In one aspect, a method is provided for making a breeding pair offertile mice, each fully derived from a donor ES cell, in the same F0generation, comprising: maintaining donor male mouse XY ES cells inculture comprising a base medium and supplements as described herein,wherein the ES cells are maintained in the base medium and supplementsfor a period of at least one day; introducing the ES cells into hostembryos (e.g., from XX mice) to form chimeric embryos; gestating thechimeric embryos in a suitable mouse to term; and, obtaining from thesuitable mouse a litter of mouse pups comprising an F0 generationfertile male XY mouse fully derived from a donor ES cell and comprisingan F0 generation fertile female XY mouse fully derived from a donor EScell.

In one embodiment, the donor ES cells comprise a genetic modification.In one embodiment, the donor ES cells comprise a genetic modificationthat is heterozygous. In one embodiment, the donor ES cells comprise aheterozygous genetic modification, the F0 generation fertile male mouseand the F0 generation fertile female XY mouse are each heterozygous forthe genetic modification, and the F0 generation fertile male and the F0generation fertile female are bred with one another and produce aprogeny that is an F1 generation mouse homozygous for the geneticmodification.

In one embodiment, the ES cells are maintained for a period of two days,three days, or four days or more.

In one aspect, a method for making a fertile female XY mouse in an F0generation is provided, comprising the steps of (a) maintaining a donorXY mouse ES cell in a medium comprising a base medium, and supplementssuitable for maintaining mouse ES cells in culture, (b) introducing thedonor XY mouse ES cell into a host embryo, (c) gestating the hostembryo, and (d) obtaining an XY female mouse progeny, wherein uponattaining sexual maturity the XY female mouse is fertile. The basemedium according to this aspect exhibits one or more characteristicsselected from (1) an osmolality of from 200 mOsm/kg to less than 329mOsm/kg, (2) a conductivity of about 11-13 mS/cm, (3) a salt of analkaline metal and a halide in a concentration of about 50-110 mM, (4) acarbonic acid salt concentration of about 17-30 mM, and (5) a totalalkaline metal halide salt and carbonic acid salt concentration of about85-130 mM.

In one embodiment, the donor XY mouse ES cell comprises a geneticmodification. In some embodiments, the genetic modification comprisesone or more of an endogenous nucleic acid sequence, a substitution ofone or more nucleic acids, a replacement of an endogenous nucleic acidsequence with a heterologous nucleic acid sequence, a knockout, and aknock-in. In one particular embodiment, the genetic modification is aknock-out of a STEAP2 gene.

In one embodiment, the base medium contains inter alia (exhibits) 50±5mM NaCl and 26±5 mM carbonate, with an osmolality of 218±22 mOsm/kg. Ina specific embodiment, the base medium exhibits about 3 mg/mL NaCl and2.2 mg/mL sodium bicarbonate, with an osmolality of about 218 mOsm/kg.

In another embodiment, the base medium exhibits 87±5 mM NaCl and 18±5mM, with an osmolality of 261±26 mOsm/kg. In a specific embodiment, thebase medium exhibits about 5.1 mg/mL NaCl and 1.5 mg/mL sodiumbicarbonate, with an osmolality of about 261 mOsm/kg.

In another embodiment, the base medium exhibits 110±5 mM NaCl and 18±5mM carbonate, with an osmolality of 294±29 mOsm/kg. In a specificembodiment, the base medium exhibits about 6.4 mg/mL NaCl and 1.5 mg/mLsodium bicarbonate, with an osmolality of about 294 mOsm/kg.

In another embodiment, the base medium exhibits 87±5 mM NaCl and 26±5 mMcarbonate, with an osmolality of about 270±27 mOsm/kg. In a specificembodiment, the base medium exhibits about 5.1 mg/mL NaCl and 2.2 mg/mLsodium bicarbonate, with an osmolality of about 270 mOsm/kg.

In another embodiment, the base medium exhibits 87±5 mM NaCl, 26±5 mMcarbonate, and 86±5 mM glucose, with an osmolality of 322±32 mOsm/kg. Ina specific embodiment, the base medium exhibits about 5.1 mg/mL NaCl,about 2.2 mg/mL sodium bicarbonate, and about 15.5 mg/mL glucose, withan osmolality of about 322 mOsm/kg.

In one aspect, a method of producing a transgenic mouse homozygous for agenetic modification in the F1 generation is provided, which comprisesthe steps of (a) crossing an F0 XY fertile female mouse producedaccording to the preceding method with a cohort F0 XY male mouse and (b)obtaining a F1 progeny mouse that is heterozygous for the geneticmodification. According to this aspect, the F0 XY fertile female mouseand the F0 XY male mouse each is heterozygous for the geneticmodification. In some embodiments, the genetic modification comprisesone or more of an endogenous nucleic acid sequence, a substitution ofone or more nucleic acids, a replacement of an endogenous nucleic acidsequence with a heterologous nucleic acid sequence, a knockout, and aknock-in.

In one specific embodiment, the F0 XY fertile female mouse is madeaccording to the preceding method in which the base medium exhibits 50±5mM NaCl and 26±5 mM carbonate, with an osmolality of 218±22 mOsm/kg. Ina particular embodiment, the base medium exhibits about 3 mg/mL NaCl and2.2 mg/mL sodium bicarbonate, with an osmolality of about 218 mOsm/kg.

In one aspect, a transgenic mouse homozygous for a genetic modification,which is produced according to the preceding method, is provided.

In one aspect, a fertile female XY mouse produced according to any ofthe preceding methods is provided. In one embodiment, the ES cells, fromwhich the XY female mouse is derived, were maintained in a base mediumthat exhibits 50±5 mM NaCl and 26±5 mM carbonate, with an osmolality of218±22 mOsm/kg. In a specific embodiment, the base medium exhibits about3 mg/mL NaCl and 2.2 mg/mL sodium bicarbonate, with an osmolality ofabout 218 mOsm/kg.

Unless expressly stated, or apparent from the context, any aspect orembodiment described herein may be combined with one another.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of breeding using F0 generation female XY micemade from different XY ES cell clones.

FIG. 2 shows generation of XY female mice from ES cells incubated withlow-salt DMEM, DMEM, or low-salt DMEM supplemented with FS(Wnt-3a-conditioned media, i.e., media conditioned by mouse L-cellstransfected with a Wnt-3a-expression construct), NaCl, and NaHCO₃.

DETAILED DESCRIPTION

All publications cited in this disclosure are hereby incorporated byreference.

The phrase “base medium” or “base media” includes a base medium known inthe art (e.g., DMEM) that is suitable for use (with added supplements)in growing or maintaining ES cells in culture. Base media suitable formaking a fertile XY female (i.e., “low-salt DMEM”) differs from basemedia typically used to maintain ES cells in culture. For purposes ofdiscussing base media in general, base media that are not suitable formaking fertile XY females are described in this section as “DMEM” and inthe following table (e.g., typical DMEM media). For purposes ofdiscussing base media suitable for making fertile XY females, the phrase“low-salt DMEM” is used. Differences between base media typically usedto maintain ES cells in culture (e.g., DMEM) and base media suitable formaking fertile XY females (e.g., “low-salt DMEM”) are articulatedherein. The phrase “low-salt DMEM” is used for convenience; suitableDMEM for making fertile XY females exhibits characteristics not limitedto “low-salt,” but includes those described herein. For example, theDMEM shown in Table 1 can be made suitable for making fertile XY femalesby altering the sodium chloride and/or sodium bicarbonate concentrationsas provided for herein, which will also result in a different osmolalityand a different conductivity as compared with the DMEM shown in Table 1.An example of base medium is Dulbeco's Modified Eagle's Medium (DMEM),in various forms (e.g., Invitrogen DMEM, Cat. No. 11971-025) (Table 1).A suitable low-salt DMEM is available commercially as KO-DMEM™(Invitrogen Cat. No. 10829-018). Base medium is typically supplementedwith a number of supplements known in the art when used to maintaincells in culture for use as donor cells. Such supplements are indicatedas “supplements” or “+supplements” in this disclosure.

TABLE 1 DMEM Base Media for Maintaining ES Cells Component mg/L mMGlycine 30 0.4 L-Arginine•HCl 84 0.398 L-Cystine•2HCl 63 0.201L-Glutamine 584 4 L-Histidine•HCl•H₂O 42 0.2 L-Isoleucine 105 0.802L-Leucine 105 0.802 L-Lysine•HCl 146 0.798 L-Methionine 30 0.201L-Phenylalanine 66 0.4 L-Serine 42 0.4 L-Threonine 95 0.798 L-Tryptophan16 0.0784 L-Tyrosine disodium salt dihydrate 104 0.398 L-Valine 94 0.803Choline chloride 4 0.0286 D-Calcium pantothenate 4 8.39 × 10⁻³ FolicAcid 4 9.07 × 10⁻³ Niacinamide 4 0.0328 Pyridoxine•HCl 4 0.0196Riboflavin 0.4 1.06 × 10⁻³ Thiamine•HCl 4 0.0119 i-Inositol 7.2 0.04Calcium Chloride (CaCl₂) (anhydrous) 200 1.8 Ferric Nitrate(Fe(NO₃)₃•9H₂O) 0.1 2.48 × 10⁻⁴ Magnesium Sulfate (MgSO₄) (anhyd.) 97.670.814 Potassium Chloride (KCl) 400 5.33 D-Glucose (Dextrose) 4500 25Phenol Red 15 0.0399 NaCl/NaHCO₃ Content of DMEM Sodium Bicarbonate(NaHCO₃) 3700 44.05 Sodium Chloride (NaCl) 6400 110.34 NaCl/NaHCO₃Content of Low-salt DMEM Sodium Bicarbonate (NaHCO₃) <3700 <44.05 SodiumChloride (NaCl) <6400 <110.34

The term “supplements” or the phrase “+supplements,” includes elementsadded to base medium for growing or maintaining donor cells in culture,e.g., for maintaining pluripotency or totipotency of donor cells inculture. For example, media supplements suitable for growing ormaintaining non-human ES cells in culture include fetal bovine serum(FBS), glutamine, penicillin and streptomycin (e.g., penstrep), pyruvatesalts (e.g., sodium pyruvate), nonessential amino acids (e.g., MEMNEAA), 2-mercaptoethanol, and LIF.

In various embodiments of media for maintaining non-human donor cells inculture, to about 500 mL of base medium the following supplements areadded: about 90 mL FBS (e.g., Hylcone FBS Cat. No. SH30070.03), about2.4 millimoles of glutamine (e.g., about 12 mL of a 200 mM glutaminesolution, e.g., Invitrogen Cat. No. 25030-081), penicillin:streptomycin(e.g., 60,000 units of Penicillin G sodium and 60 mg of streptomycinsulfate, with about 51 mg of NaCl; e.g., about 6 mL of Invitrogenpennstrep, Cat. No. 15140-122), about 0.6 millimoles of sodium pyruvate(e.g., 6 mL of 100 mM sodium pyruvate, Invitrogen Cat. No. 11360-070),about 0.06 millimoles of nonessential amino acids (e.g., about 6 mL ofMEM NEAA, e.g., MEM NEAA from Invitrogen Cat. No. 11140-050), about 1.2mL 2-mercaptoethanol, and about 1.2 micrograms of LIF (e.g., about 120microliters of a 10⁶ units/mL LIF preparation; e.g., about 120microliters of Millipore ESGRO™-LIF, Cat. No. ESG1107). When composingbase media for maintaining XY ES cells for making fertile XY females,typically the same supplements in about the same amounts are employed,but the composition of the base medium will differ (from DMEM, e.g.,from the medium described in the table above) and the difference(s)correspond to the difference(s) taught herein.

In some embodiments, supplements include Wnt-conditioned media, e.g.,Wnt-3a conditioned media.

The term “animal,” in reference to donor cells and/or host embryos,includes mammals, fishes, and birds. Mammals include, e.g., humans,non-human primates, rodents (e.g., mice, rats, hamsters, guinea pigs),livestock (e.g., bovine species, e.g., cows, steer, etc.; ovine species,e.g., sheep, goats, etc.; and porcine species, e.g., pigs and boars).Birds include, e.g., chickens, turkeys, ostrich, geese, ducks, etc. Thephrase “non-human animal,” in reference to donor cells and/or hostembryos, excludes humans.

In various embodiments, the donor cell and/or the host embryo are notfrom one or more of the following: Akodon spp., Myopus spp., Microtusspp., Talpa spp. In various embodiments, the donor cell and/or the hostembryo are not from any species of which a normal wild-typecharacteristic is XY female fertility. In various embodiments, where agenetic modification is present in the donor cell or the host embryo,the genetic modification is not an XYY or XXY, a Tdy-negative sexreversal, Tdy-positive sex reversal, an X0 modification, an aneuploidy,an SRY translocation or modification, an fgf^(−/−) genotype, or a SOX9modification.

Overview

Methods for making nonhuman animals, e.g., mice, from donor ES cells andhost embryos are known in the art. Donor ES cells are selected forcertain characteristics that enhance the ability of the cells topopulate a host embryo and thus contribute in part or in substantialpart to an animal formed by the donor ES cells and the host embryo. Theanimal formed may be male or female, based in large part on the genotypeof the ES cell (e.g., XY or XX).

The majority of ES cell liens for making mice have a male XY genotype.Because of the dominance of the Y chromosome in mammalian sexdetermination, XY ES cells, when introduced into a host embryo andgestated, nearly always result in the first generation (F0) inphenotypically male animals that are chimeras, i.e., that contain cellsderived from the male donor ES cell (XY) and cells derived from the hostembryo, which can be either male (XY) or female (XX). To the extent thatphenotypic females are observed in the F0 generation, these typicallyarise from the introduction of XY ES cells into a female XX embryo thatresults in a chimera whose ES cell contribution is insufficient tomasculinize the embryonic genital ridge. In most cases such femalechimeras do not produce oocytes derived from the XY ES cells and,therefore, are not capable of transmitting the ES cell genome to thenext generation. In rare cases, female chimeras do not produce oocytesderived from the XY ES cells; these females can transmit the ES cellgenome to the next generation (see, e.g., Bronson et al. (1995) Highincidence of XXY and XYY males among the offspring of female chimerasfrom embryonic stem cells, Proc. Natl. Acad. Sci USA 92:3120-3123).

Phenotypically female mice with an XY genotype can arise as the resultspecific mutations. See, e.g., Lovell-Badge et al. (1990) XY female miceresulting from a heritable mutation in the primary testis determininggene, Tdy, Development 109:635-646; see also, Colvin et al. (2001)Male-to-Female Sex Reversal in Mice Lacking Fibroblast Growth Factor 9,Cell 104(6):875-889 (Fgf−/−XY females that die at birth from lunghypoplasia). The South American Akadon spp. of rodents comprise XYfemales (see, e.g., Hoekstra et al. (2000) Multiple origins of XY femalemice (genus Akodon): phylogenetic and chromosomal evidence, Proc. R.Soc. Lond. B 267:1825-1831), but ES cell lines from such mice aregenerally not available and not widely used, if at all.

In some instances, e.g., using the VELOCIMOUSE® method (see, e.g., U.S.Pat. Nos. 7,659,442, 7,576,259, 7,294,754, and Poueymirou et al. (2007)F0 generation mice fully derived from gene-targeted embryonic stem cellsallowing immediate phenotypic analyses, Nat. Biotech. 25(1):91-99; eachhereby incorporated by reference), it is possible to obtain F0generation mice that are fully derived from the donor ES cell. Undernormal circumstances and standard experimental conditions, XY donor EScells produce only phenotypically male fully ES cell-derived mice, whileES cells that are XX or X) (XY ES cells that have lost the Y chromosome)produce only phenotypically female fully ES cell-derived mice. Toproduce mice with homozygous targeted mutations from the male and femalefully ES cern-derived mice requires two subsequent generations ofbreeding to first produce the F1 generation heterozygous male andfemales that when intercrossed have the potential to produce homozygousprogeny in the F2 generation.

The inventors have devised a method for making a phenotypically femalefertile XY mouse from an XY donor cell (e.g., an XY donor cell derivedfrom a phenotypically male mouse) and a suitable host embryo. The methodcomprises making such a mouse in the F0 generation, which allows forforming a breeding pair (a male F0 and a female F0) in the F0generation. This is particularly useful where the donor cell comprises aheterozygous genetic modification, and a homozygous mouse is desired.Although this disclosure illustrates the invention in the context ofmaking phenotypically female fertile XY mice from donor mouse XY EScells, the methods and compositions described herein may be applied tomake phenotypically female XY fertile nonhuman animals from any suitablenonhuman cell (e.g., an iPS cell, an ES cell, or a pluripotent cell) andany suitable nonhuman embryo.

Methods and compositions are described that include conditions formaintaining a donor cell such that when the donor cell is used togenerate an animal by introducing the donor cell into a host embryo, theanimal so generated includes a phenotypically female fertile XY animal.A phenotypically female fertile XY animal includes an animal thatexhibits sufficient phenotypically female characteristics to ovulate andto gestate an embryo upon fertilization of an ovum produced by ovulationin the animal, including to gestate an embryo to term and give birth toa live-born animal.

The inventors have devised a method that results, in various embodimentsat least about 10%, 15%, 20%, or 25% or more of the time, in birth of afertile female XY mouse from an XY mouse ES cell.

Animal Husbandry

In one aspect, a method is provided for generating a female animal froma sperm cell and an egg cell, comprising maintaining the sperm celland/or the egg cell in a medium comprising low-salt base medium for one,two, three, or four or more days prior to fertilization, contacting thesperm cell and the egg cell under conditions that permit fertilizationto form a fertilized egg, implanting the fertilized egg in a suitablehost for gestation, gestating in the host, and obtaining a littercomprising a female animal.

In one embodiment, the fertilized egg is further maintained in themedium comprising low-salt base medium for one, two, three, or four ormore days prior to implantation in the suitable host.

In one aspect, a method is provided for favoring the generation of afemale animal from a fertilized egg or an embryo, comprising maintainingthe fertilized egg or embryo in a medium comprising low-salt base mediumfor one, two, three, or four or more days prior to implantation in asuitable host, implanting the fertilized egg or embryo into a suitablehost for gestation, gestating the fertilized egg or embryo in the host,and obtaining a litter comprising a female animal.

In one aspect, the methods and compositions of the invention areemployed to make a female pet, a female domesticated farm animal, afemale animal as a scientific research subject, or an animal of anendangered species. In one embodiment, the animal is a mouse, rat,hamster, monkey, ape, cat, dog, cow, horse, bull, sheep, goat, pig,deer, and bison.

EXAMPLES Example 1 Donor XY ES Cells and Host Embryos

Donor Cells and Host Embryos.

Donor ES cells were 129S6C57Bl6/F1 hybrid ES cells. The donor ES cellswere frozen in freezing medium containing 10% DMSO until use. Oncethawed, donor ES cells were maintained in base medium and supplements asdescribed below. Host embryos were from Swiss Webster (SW) mice, andwere maintained in KSOM medium (Millipore) until use. Eight-cell embryoswere obtained as previously described (Poueymirou et al. (2007) NatureBiotech. 25(1):91-99; U.S. Pat. Nos. 7,659,442, 7.576,259, and7,294,754).

DMEM ES cells: ES cells prepared and frozen in DMEM were thawed in DMEM,grown for three days, and microinjected into host embryos in DMEM.

Low-salt DMEM ES cells: ES cells prepared and frozen in low-salt DMEMwere thawed in low-salt DMEM (KO-DMEM), grown for three days, andmicroinjected into host embryos in DMEM.

FS low-salt DMEM: ES cells prepared and frozen in low-salt DMEM werethawed and maintained in low-salt DMEM (440 mL)+10% Wnt-3a-conditionedmedia (FS) (60 mL), and microinjected into host embryos in DMEM.

Low-salt DMEM+NaCl+NaHCO₃: ES cells prepared and frozen in low-salt DMEMwith added NaCl (1,300 mg/L) and NaHCO₃ (1,500 mg/L) and microinjectedinto host embryos in DMEM.

10% Wnt-3a-conditioned media (FS): Wnt-3a-conditioned media was madefrom cultures of mouse L cells transformed with a Wnt-3a expressionvector (ATCC CRL-2647). The L cells are grown according to ATCCinstructions (except that KO-DMEM™ is used in place of DMEM), in aFibraStage™ (New Brunswick) system.

Example 2 Making F0 Generation Mice Derived from Donor ES Cells

Generating F0 Generation Mice.

Donor ES cells were introduced into 8-cell stage pre-morula host embryosusing the VELOCIMOUSE® method, as described previously (Poueymirou etal. (2007) Nature Biotech. 25(1):91-99; U.S. Pat. Nos. 7,659,442,7.576,259, and 7,294,754), except that the mouse ES cells weremaintained in the base medium plus supplements as described herein. Formicroinjection, ES cells were grown and microinjected into the embryos,and the embryos were cultured overnight in either KSOM or DMEM mediumprior to implantation into surrogate mothers.

Example 3 F0 Generation Fertile Female Mice from Donor XY ES Cells

In a typical protocol, ES cells are thawed in the presence of KO-DMEM™and grown for one passage (about 5 five days). Passaged cells are thenelectroporated with a gene targeting vector and then placed underselection for 10 days in a medium comprising KO-DMEM™ (Invitrogen Cat.No. 10829-018). Drug-resistant cells are harvested and expanded in amedium comprising KO-DMEM™, then frozen. For microinjection, cells arethawed in KO-DMEM™ and grown for 3 days in KO-DMEM™, then microinjectedinto embryos in DMEM. The embryos are then introduced into surrogatemothers for gestation.

Mouse pups were initially characterized as male or female based on theappearance of external genitalia in order to select breeding pairs.

FIG. 1 shows that F0 XY females exhibit a high rate of fertility.Twenty-one out of 33 F0 XY females produced litters.

Example 4 Comparing DMEM with Low-salt DMEM

Osmolality was measured on a Advanced® Model 3250 Single-SampleOsmometer. Conductivity was measured on a Mettler Toledo GmbHSevenMulti™ ECN #15055 conductivity meter.

The effect of low-salt DMEM and of DMEM (each with supplements) on theformation of F0 generation XY females from XY ES cells was studied.Table 2 shows the osmolality and conductivity values of base media withand without additional salts and/or supplements. The indicator“+supplements”=addition (to 0.5 L of base medium) of the following: 90mL Hyclone FBS (Cat. No. SH30070.03), 12 mL of Invitrogen glutaminesolution (Cat. No. 25030-081), 6 mL of Invitrogen Pen Strep (Cat. No.15140-122), 6 mL of Invitrogen sodium pyruvate (Cat. No. 11360-070), 6mL of MEM NEAA (Invitrogen Cat. No. 11140-050), 1.2 mL2-mercaptoethanol, and 120 microliters of Millipore ESGRO™-LIF (Cat. No.ESG1107).

FIG. 2 shows a comparison of XY ES cells grown in different media priorto microinjections into host embryos. XY ES cells grown and maintainedin low-salt DMEM and then injected into embryos produced XY females. XYES cells grown and maintained in low-salt DMEM supplemented with NaCland NaHCO₃ and then injected into embryos produced no XY females. Thisdemonstrates that XY female production is promoted when XY ES cells aremaintained in low-salt DMEM, and that the sex ratio of XY ES cells canbe controlled by altering the salt concentration of the base medium.Adding a Wnt-3a-conditioned medium (10% FS) to a low-salt DMEM increasedthe frequency of production of F0 XY females.

Furthermore, the efficiency of generating ES cell-derived mice in the F0increased when the ES cells were maintained in Low-salt DMEM. The ratioof ES cell-derived pups to total pups generated in the F0 generationincreased from about 23% for ES cells maintained in DMEM, to 61% for EScells maintained in low-salt DMEM, to 72% for ES cells maintained inlow-salt DMEM supplemented with 10% Wnt-3a-conditioned media. See FIG.2.

TABLE 2 Comparison of DMEM and Low-salt DMEM Physical CharacteristicsConductivity Osmolality Medium for ES Cells (mS/cm) (mOsm/kg) Low-saltDMEM, alone 12.84 270 DMEM, alone 15.40 337 Low-salt DMEM + NaHCO₃ +NaCl, alone 15.82 342 Low-salt DMEM, + supplements 12.75 279 DMEM, +supplements 14.91 330 Low-salt DMEM + NaHCO₃ + NaCl, + 15.29 335supplements

Example 5 Analysis of F0 Generation Mice

Coat Color.

Mice were analyzed for coat color contribution from donor XY ES cells(agouti) and host embryo (white). None of the F0 generation miceexhibited any coat color contribution from host embryos.

Gender.

F0 generation pups were identified as female or male by visualinspection of the external genitalia. F0 pups were assigned gender andpaired for breeding based on visual inspection.

Genotyping.

The presence of an X chromosome was detected using a TAQMAN™ QPCR assayspecific for a sequence on the X chromosome. The presence of Ychromosome was detected using a TAQMAN™ QPCR assay specific for asequence on the Y chromosome. The genotyping of phenotypically female F0generation mice indicated a single copy of the X chromosome and a singlecopy of the Y chromosome in those phenotypically female mice tested.

Karyotyping.

Six F0 generation XY females were karyotyped. Karyotyping resultsindicated that all six had a normal X and a normal Y chromosome.

XY Female Reproductive Anatomy.

Several F0 generation XY females were examined for internal reproductiveorgans. All of the F0 XY females examined appeared to have normal femaleinternal reproductive organs. Tissue samples from each reproductiveorgan (ovary, oviduct, uterus) were genotyped, and the results indicatedthat the tissues had a uniform XY genotype.

Example 6 Analysis of the Effect of Osmolality on Efficiency ofGenerating ES Cell-Derived Pups and XY Females

To determine the effect of osmolality on the generation of XY femalesfrom XY ES cells maintained in low-salt, low-carbonate DMEM, glucose wasadded to low-salt, low-carbonate DMEM to bring the osmolality to withinthat of DMEM. Osmolality was measured on a Advanced® Model 3250Single-Sample Osmometer.

Donor XY ES cells were maintained in low-salt, low-carbonate, highglucose DMEM containing inter alia 5.1 mg/ml NaCl, 2.2 mg/ml NaHCO₃, and15.5 mg/ml glucose, having an osmolality of 322 mOsm/kg(“DMEM-LS/LC/HG”). Upon transfer of said ES cells into embryos per theVELOCIMOUSE® method (supra), 15% of all resultant ES cell-derived F0progeny were phenotypically female XY mice. As a negative control, inthe F0 generation, no phenotypically female XY mice were derived from EScells maintained in DMEM (“DMEM”: 6.4 mg/ml NaCl, 3.7 mg/ml NaHCO₃, and4.5 mg/ml glucose; 329 mOsm/kg). This 15% F0 XY female result liesbetween the 0% F0 XY females from DMEM-derived ES cells (329 mOsm/L) andthe 27.8% F0 XY female mice derived from ES cells maintained inlow-salt, low carbonate DMEM (“DMEM-LS/LC”: 5.1 mg/ml NaCl, 2.2 mg/mlNaHCO₃, and 4.5 mg/ml glucose; 270 mOsm/kg). Thus, one interpretation isthat osmolality provides some of the feminization effect, but not all.An alternative explanation is that the low salt and/or low carbonateprovides the feminization effect, and high glucose impedes to someextent the feminization of XY ES cells. See Table 3.

Furthermore, the efficiency of generating ES cell-derived mice (Table 3)in F0 when the ES cells were maintained in DMEM-LS/LC/HG (i.e., about40%) was greater than that for ES cells maintained in DMEM (i.e., about22%), but not quite as high as that for ES cells maintained inDMEM-LS/LC (i.e., about 51%). See Table 3.

TABLE 3 Effect of Osmolarity, Salt, and Carbonate on ES-cell DerivedPups and F0 XY Females ES- Osmolality NaCl NaHCO₃ Glucose Derived pups/ES-derived pups Media (mOsm/kg) (mg/mL) (mg/mL) (mg/mL) Total pups XYmale XY female DMEM 329 6.4 3.7 4.5 13/58 13/13  0/13 (22.4%)   (0%)DMEM- 270 5.1 2.2 4.5 36/71 26/36 10/36 LS/LC (50.7%) (27.8%) DMEM- 3225.1 2.2 15.5 20/50 17/20  3/20 LS/LC/HG  (40%)  (15%) DMEM- 218 3.0 2.24.5 53/58 35/53 18/53 VLS/LC (91.4%) (34.0%) DMEM- 261 5.1 1.5 4.5 50/5733/50 17/50 LS/VLC (87.7%)  (34%) DMEM- 294 6.4 1.5 4.5 49/68 35/4914/49 VLC (72.1%) (28.6%)

Example 7 Analysis of the Effect of Salt Concentration on Efficiency ofGenerating ES cell-Derived Pups and XY Females

To determine the effect of salt concentration or ionic strength on thegeneration of XY females from XY ES cells, ES cells were maintained invery low salt (DMEM-VLS/LC: 3.0 mg/mL NaCl, 2.2 mg/mL NaHCO3, 4.5 mg/mLglucose, at 218 mOsm/kg). Upon transfer of said ES cells into embryosper the VELOCIMOUSE® method (supra), 34% of all resultant EScell-derived F0 progeny were phenotypically female XY mice; a slightincrease over the DMEM-LS/LC control level of 27.8%. Interestingly,91.4% of the F0 pups resulting from the transfer of ES cells maintainedin DMEM-VLS/LC media were ES cell-derived; whereas only 50.7% and 22.4%were ES cell-derived in the DMEM-LS/LC and DMEM controls, respectively.

In another experiment, ES cells were maintained in high salt and lowcarbonate media (DMEM-HS/VLC: 6.4 mg/mL NaCl, 1.5 mg/mL NaHCO3, 4.5mg/mL glucose, at 294 mOsm/kg). Upon transfer of said ES cells intoembryos per the VELOCIMOUSE® method (supra), 28.6% of all resultant EScell-derived F0 progeny were phenotypically female XY mice; a slightincrease over the DMEM-LS/LC control level of 27.8%. Interestingly,72.1% of the F0 pups resulting from the transfer of ES cells maintainedin DMEM-HS/VLC media were ES cell-derived; whereas only 50.7% and 22.4%were ES cell-derived in the DMEM-LS/LC and DMEM controls, respectively.

These results confirm that low salt and/or low carbonate contribute bothto the increase in proportion of ES cell-derived F0 progeny as well asF0 XY females. (See Table 3.)

Example 8 Analysis of the Effect of Carbonate Concentration onEfficiency of Generating ES Cell-Derived Pups and XY Females

To determine the effect of carbonate concentration on the generation ofXY females from XY ES cells, ES cells were maintained in low salt andvery low carbonate media (DMEM-LS/VLC: 5.1 mg/mL NaCl, 1.5 mg/mL NaHCO3,4.5 mg/mL glucose, at 261 mOsm/kg). Upon transfer of said ES cells intoembryos per the VELOCIMOUSE® method (supra), 34% of all resultant EScell-derived F0 progeny were phenotypically female XY mice; a slightincrease over the DMEM-LS/LC control level of 27.8%. Interestingly,87.7% of the F0 pups resulting from the transfer of ES cells maintainedin DMEM-LS/VLC media were ES cell-derived; whereas only 50.7% and 22.4%were ES cell-derived in the DMEM-LS/LC and DMEM controls, respectively.

These results confirm that low carbonate contributes both to theincrease in proportion of ES cell-derived F0 progeny as well as F0 XYfemales. (See Table 3.)

Example 9 Phenotype of F0 XY Female Mice

F0 XY phenotypic female mice exhibited relatively normal phenotypeattributes compared to F1 XX phenotypic female mice of the same strain.The XY female mice however did exhibit a larger range of values for eachphysical parameter. The body weight of the adult XY females ranged fromabout 15 grams to about 30 grams with an average of about 21.5 grams.The body weight of the adult XX females ranged from about 16 grams toabout 17 grams with an average of about 16.8 grams.

The ratio of the distance between the anus and the genitals wasdetermined and calculated as a ratio of body mass (anogenital distance(cm)/body mass (g)). The ratio for F0 XY females ranged from about 0.11cm/g to about 0.24 cm/g with an average of about 0.16 cm/g. The ratiofor F1 XX females ranged from about 0.17 cm/g to about 0.19 cm/g with anaverage of about 0.18 cm/g.

There was no significant difference between the relative masses ofvarious organs (e.g., liver, kidneys, heart and lung, and spleen) forthe XY female mice and the XX female mice. Relative masses are expressedas organ mass (mg)/body mass (g). The relative mass of the liver of theF0 XO females ranged from about 35 mg/g to about 50 mg/g with an averageof about 42 mg/g. The relative mass of the liver of the F1 XX femalesranged from about 37.5 mg/g to about 46.9 mg/g with an average of about42.5 mg/g. The relative mass of the kidneys of the F0 XO females rangedfrom about 11.5 mg/g to about 15 mg/g with an average of about 13.4mg/g. The relative mass of the kidneys of the F1 XX females ranged fromabout 12.6 mg/g to about 13.8 mg/g with an average of about 13.7 mg/g.The relative combined mass of the heart and lungs of the F0 XO femalesranged from about 14.3 mg/g to about 18.9 mg/g with an average of about16.1 mg/g. The relative combined mass of the heart and lungs of the F1XX females ranged from about 14.7 mg/g to about 16.1 mg/g with anaverage of about 15.9 mg/g. The relative mass of the spleen of the F0 XOfemales ranged from about 2.7 mg/g to about 6.6 mg/g with an average ofabout 3.3 mg/g. The relative mass of the spleen of the F1 XX femalesranged from about 2.7 mg/g to about 4.0 mg/g with an average of about3.8 mg/g.

The F0 XY female mice were shown to have relatively normal serum levelsof electrolytes, enzymes, glucose, proteins, lipids and other indiciacompared to syngeneic F1 XX females. The XY female mice however didexhibit a larger range of values for each measured serum parameter. Theserum sodium levels of the adult XY females ranged from about 150 mEq/Lto about 159 mEq/L; and the levels for the XX females ranged from about148 mEq/L to about 155 mEq/L.

The serum potassium levels of the adult XY females ranged from about 0.7mEq/L to about 7 mEq/L; and the levels for the XX females were about 0.7mEq/L.

The serum chloride levels of the adult XY females ranged from about 111mEq/L to about 121 mEq/L; and the levels for the XX females ranged fromabout 113 mEq/L to about 120 mEq/L.

The serum calcium levels of the adult XY females ranged from about 7mEq/L to about 9 mEq/L; and the levels for the XX females were about 7mEq/L.

The serum alkaline phosphatase levels of the adult XY females rangedfrom about 124 U/L to about 285 U/L; and the levels for the XX femalesranged from about 191 U/L to about 236 U/L.

The serum alanine aminotransferase levels of the adult XY females rangedfrom about 21 U/L to about 285 U/L; and the levels for the XX femalesranged from about 13 U/L to about 34 U/L.

The serum aspartate aminotransferase levels of the adult XY femalesranged from about 42 U/L to about 190 U/L; and the levels for the XXfemales ranged from about 42 U/L to about 269 U/L.

The serum lipase levels of the adult XY females ranged from about 16 U/Lto about 49 U/L; and the levels for the XX females ranged from about 21U/L to about 26 U/L.

The serum glucose levels of the adult XY females ranged from about 227mg/dL to about 319 mg/dL; and the levels for the XX females ranged fromabout 255 mg/dL to about 270 mg/dL.

The total serum protein levels of the adult XY females ranged from about4.6 mg/dL to about 5.2 mg/dL; and the levels for the XX females rangedfrom about 4.6 mg/dL to about 4.8 mg/dL.

The serum albumin levels of the adult XY females ranged from about 3mg/dL to about 3.5 mg/dL; and the levels for the XX females ranged fromabout 3.1 mg/dL to about 3.2 mg/dL.

The serum cholesterol (total) levels of the adult XY females ranged fromabout 58 mg/dL to about 108 mg/dL; and the levels for the XX femalesranged from about 61 mg/dL to about 85 mg/dL.

The serum triglyceride levels of the adult XY females ranged from about42 mg/dL to about 89 mg/dL; and the levels for the XX females rangedfrom about 39 mg/dL to about 48 mg/dL.

The serum HDL levels of the adult XY females ranged from about 29 mg/dLto about 57 mg/dL; and the levels for the XX females ranged from about23 mg/dL to about 42 mg/dL.

The serum LDL levels of the adult XY females ranged from about 3.7 mg/dLto about 11 mg/dL; and the levels for the XX females ranged from about3.7 mg/dL to about 13 mg/dL.

The blood urea nitrogen (BUN) levels of the adult XY females ranged fromabout 12 mg/dL to about 27 mg/dL; and the levels for the XX femalesranged from about 18 mg/dL to about 21 mg/dL.

The serum magnesium levels of the adult XY females ranged from about 1.6mg/dL to about 3.2 mg/dL; and the levels for the XX females were about2.1 mg/dL.

The serum inorganic phosphate levels of the adult XY females ranged fromabout 5.1 mg/dL to about 10 mg/dL; and the levels for the XX femalesranged from about 7.2 mg/dL to about 8.4 mg/dL.

The serum uric acid levels of the adult XY females ranged from about 0.9mg/dL to about 3.5 mg/dL; and the levels for the XX females ranged fromabout 0.7 mg/dL to about 2.2 mg/dL.

Example 10 Production of Homozygous Genetically Modified Mouse in the F1Generation

To determine whether F1 mice homozygous for a genetic modification couldbe made, F0 XY female mice containing at least one knocked-out allele ofa STEAP2 gene was mated to XY male cohort containing the same STEAP2gene knock-out. (The STEAP2 (Six transmembrane epithelial antigen of theprostate 2) gene encodes for a putative 6 membrane metalloreductase withferrireductase and cupric reductase activity, and has been shown tostimulate the cellular uptake of both iron and copper in vitro. As acell-surface antigen, STEAP2 is a potential diagnostic or therapeutictarget in prostate cancer. STEAP2 was significantly elevated in bothuntreated primary and hormone-refractory prostate carcinomas than inbenign prostate hyperplasias, suggesting that it may be involved in thedevelopment of prostate cancer. STEAP2 KO mouse has not been reported.See Ohgami et al., BLOOD, vol. 108(4):1388-1394, 2006.) The results aredepicted in Table 4.

TABLE 4 Genotypes of STEAP2 F1 Cohorts from F0 XY Males × F0 XY FemalesSex Sex Chromosomes STEAP2 Genotype (N/%) Phenotype (N/%) Wt/wt Wt/KOKO/KO Female XX (7/15%) 2/4.3% 3/6.4% 2/4.3% XO (4/8.5%) 1/2.1% 1/2.1%2/4.3% XY (0/0%) 0/0% 0/0% 0/0% Male XY (17/36%) 6/12.8% 6/12.8% 5/10.6%XXY (8/17%) 3/6.4% 3/6.4% 2/4.3% XYY (11/23.5%) 3/6.4% 3/6.4% 5/10.6%

We claim:
 1. A method for making and breeding a fertile female XY mousein an F0 generation, comprising: (a) maintaining donor XY mouse ES cellsin a medium comprising (i) a base medium, and (ii) supplements suitablefor growing the mouse ES cells in culture and maintaining pluripotency,wherein the donor XY mouse ES cells do not comprise a Tdy-negative sexreversal genetic modification and wherein the base medium comprises anosmolality of 218 mOsm/kg to 322 mOsm/kg, a sodium chloride (NaCl)concentration of 50-110 mM and a sodium bicarbonate concentration of13-25 mM; (b) introducing a donor XY mouse ES cell from step (a) into apre-morula stage host mouse embryo; (c) introducing the host mouseembryo of step (b) into a recipient female mouse and gestating the hostmouse embryo; (d) obtaining an F0 XY mouse progeny comprising a F0 XYphenotypically female mouse, wherein upon attaining sexual maturity theF0 XY female mouse is fertile; and (e) breeding the F0 XY fertile femalemouse to produce progeny.
 2. The method of claim 1, wherein the donor XYmouse ES cell comprises a genetic modification.
 3. The method of claim2, wherein the genetic modification comprises a substitution of one ormore nucleic acids.
 4. The method of claim 2, wherein the geneticmodification is a knock-out of a STEAP2 gene.
 5. The method of claim 1,wherein the base medium comprises 87±5 mM NaCl, 18±5 mM sodiumbicarbonate, and 261±26 mOsm/kg.
 6. The method of claim 1, wherein atleast 34% of the F0 XY mouse progeny derived from the donor XY mouse EScell are phenotypically female XY mice.
 7. The method of claim 1,wherein at least 15% of the F0 XY mouse progeny derived from the donorXY mouse ES cell are phenotypically female XY mice.
 8. The method ofclaim 1, wherein all of the females derived from the donor XY mouse EScell in the F0 generation have a XY genotype.
 9. The method of claim 1,wherein the breeding comprises crossing the F0 XY fertile female mousewith a cohort F0 XY male mouse, wherein the F0 XY fertile female mouseand the F0 XY male mouse each is heterozygous for a geneticmodification, and obtaining an F1 progeny mouse that is homozygous forthe genetic modification.